www.nature.com/scientificreports OPEN Genomic structure analysis of a set of Oryza nivara introgression lines and identification of yield- Received: 08 December 2015 Accepted: 19 May 2016 associated QTLs using whole- Published: 02 June 2016 genome resequencing Xin Ma1, Yongcai Fu1, Xinhui Zhao1,2, Liyun Jiang1, Zuofeng Zhu1, Ping Gu1, Wenying Xu2, Zhen Su2, Chuanqing Sun1,2 & Lubin Tan1 Oryza nivara, an annual wild AA-genome species of rice, is an important gene pool for broadening the genetic diversity of cultivated rice (O. sativa L.). Towards identifying and utilizing favourable alleles from O. nivara, we developed a set of introgression lines (ILs) by introducing O. nivara segments into the elite indica rice variety 93-11 background through advanced backcrossing and repeated selfing. Using whole-genome resequencing, a high-density genetic map containing 1,070 bin-markers was constructed for the 131 ILs, with an average length of 349 kb per bin. The 131 ILs cover 95% of O. nivara genome, providing a relatively complete genomic library for introgressing O. nivara alleles for trait improvement. Using this high-density bin-map, QTL mapping for 13 yield-related traits was performed and a total of 65 QTLs were detected across two environments. At ~36.9% of detected QTLs, the alleles from O. nivara conferred improving effects on yield-associated traits. Six cloned genes,Sh4 /SHA1, Bh4, Sd1, TE/TAD1, GS3 and FZP, colocalised in the peak intervals of 9 QTLs. In conclusion, we developed new genetic materials for exploration and use of beneficial alleles from wild rice and provided a basis for future fine mapping and cloning of the favourableO. nivara-derived QTLs. Cultivated rice (Oryza sativa L.) is the staple food for billions of people worldwide and its production is essential for global food security. Utilization of heterosis and agronomically important genes has led to great progress in improving rice production during the past decades1,2. However, the sustainable increase of rice yield faces the challenges of global population explosion, rapid climatic change and shortages of water and land3,4. A key limiting factor for the long-term and sustained improvement of modern rice cultivars is genetic diversity, which has mark- edly decreased by strong artificial selection5,6. Discovery and deployment of favourable alleles from wild rice has been proposed as a key strategy for broadening the genetic diversity of cultivated rice and bringing a new green revolution in rice breeding. Thousands of wild relatives of rice have been collected and are stored in germplasm banks. These germplasms offer a wide range of genetic resources for creating superior rice varieties in the future. However, it is difficult to utilize wild rice directly because of its overall inferior performance. The advanced backcross method is an effective strategy for identifying and introducing agronomically beneficial alleles from unadapted germplasm into the cultivated gene pool7. Following this strategy, many favourable alleles for yield-associated traits from O. rufipogon, a perennial wild rice, have been successfully identified. For example, some advanced backcross popu- lations using the same O. rufipogon accession (IRGC 105491) as the donor parent and different O. sativa cultivar as the recurrent parent were developed and several valuable QTLs, of which alleles from the wild rice enhanced yield and yield components, were identified8–12. Additional researches have further confirmed that some QTLs 1National Center for Evaluation of Agricultural Wild Plants (Rice), Beijing Key Laboratory of Crop Genetic Improvement, Laboratory of Crop Heterosis and Utilization, MOE, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. 2State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100193, China. Correspondence and requests for materials should be addressed to L.T. (email: [email protected]) SCIENTIFIC REPORTS | 6:27425 | DOI: 10.1038/srep27425 1 www.nature.com/scientificreports/ truly improved the yield performance of elite cultivar or conferred beneficial effect to enhance the trait value of the breeding lines12,13. Many O. rufipogon introgression lines (ILs) have been developed by repeated selfing of advanced backcross populations and provide ideal genetic materials for evaluating agronomic traits and for positional cloning of interesting genes or QTLs, because of the simple and stable genetic background of wild rice ILs14–18. The annual diploid species O. nivara is another close wild relative of cultivated rice with great potential to improve elite cultivars, for instance, enhanced grassy stunt virus resistance and yield components has been achieved through transferring useful genes from O. nivara into cultivated rice varieties19–21. However, only few introgression libraries have been developed with O. nivara. The rapid development of next-generation sequencing (NGS) technology has opened new opportunities for dissecting complex traits22,23. Compared with QTL mapping using conventional molecular markers, the whole-genome sequencing approach has more advantages, including the construction of a high-density genetic map based on high quality SNP markers, reduction of the amount of time and effort required for QTL mapping, and improving the accuracy and precision of QTL mapping for the complex traits. Using a whole-genome rese- quencing strategy, Huang et al.24 constructed a high-resolution bin map that delimited the largest effect QTL for plant height (PH) into a 100 kb physical interval. Wang et al.25 identified 49 loci associated with 14 agronomic traits using this map, of which 5 large-effect loci were mapped to relatively small chromosomal regions containing 5 strong candidate genes. Owing to its high resolution, accuracy and low cost, this strategy has also been widely applied in other crop plants, such as maize26,27, sorghum28 and soybean29. The combination of NGS and ILs pro- vides a new strategy for exploiting the untapped reserve of wild alleles associated with agronomic traits. In the present study, an IL population was constructed with the elite indica variety 93-11 as the recipient and O. nivara accession W2014 as the donor. Both parents and ILs were whole-genome resequenced to characterise the genomic structure of each IL. Using the high-density genetic map incorporating 1,070 bin-markers, a total of 65 QTLs for 13 yield-associated traits were detected, with favourable alleles from W2014 at 24 of these QTLs. Our finding indicated that the new set of ILs offers a unique opportunity for exploring and utilizing beneficial alleles from wild rice O. nivara. Results Genomic structure analysis of O. nivara ILs. Identification of SNPs of parents and 131 ILs. With respect to the reference genome (Os-Nipponbare-Reference-IRGSP-1.0, MSU release 7), the parents 93-11 and W2014 were resequenced with an estimated sequence depth of 13.55X and 13.64X, respectively, and the ILs with a mean depth of 2.83X. Using SAMtools software30, a total of 181,957 high-quality SNPs were identified between the two parents, with an average of 4.9 SNPs per 10 kb (Supplementary Dataset 1 and Supplementary Figure 1). Of these SNPs, 92,127 were located in the genic regions (Supplementary Table 1). Subsequently, we performed variant effect prediction analysis using SnpEff software31, and found that a total of 997 SNPs were predicted with high impact effect for protein sequences, either by altering the start codon, splice sites, or stop codon (Supplementary Dataset 2). Construction of the bin map. Following a previously reported procedure24, the genome-wide graphical geno- types of 131 ILs were identified. Based on the analysis of recombination breakpoints, a high-density bin map consisting of 1,070 bin markers was constructed (Supplementary Dataset 3). The average physical length of a bin was 349 kb, ranging from 5.0 kb to 7.37 Mb (Fig. 1a). The bins near centromeric regions were generally with larger length than those in the distal regions of chromosomes, which is expected because of the varied recombination rates in chromosome (Fig. 1b). On an average, each bin harboured 53 genes, and 324 (30.28%) bins contained fewer than 10 genes (Fig. 1c), indicating that the resolution of a bin map is much higher than that of a conven- tional map made using PCR-based markers. In the IL population, a total of 1,164 crossover events (COs) were detected with an average of 8.89 per IL (Supplementary Figure 2). There was a linear correlation between the length of chromosomes and the average number of COs per chromosome (Supplementary Figure 2). Number, length and distribution of the introgressed segments. The 131 ILs carried a total of 767 chromosomal segments from O. nivara in the genetic background of 93-11, including 593 homozygous and 174 heterozygous segments (Supplementary Table 2). On an average, each IL carried 5.8 introgressed segments with from 1 to 12 for homozygous segment and from 0 to 4 for heterozygous (Fig. 2, Supplementary Table 2). Forty-seven lines carried only homozygous segments. Additionally, we observed an uneven distribution of introgressed segments among the 12 chromosomes (Fig. 3). The long arms of chromosome 1 (physical interval: 32–42 Mb), chromo- some 2 (26–33 Mb), and the short arm of chromosome 9 (0–10 Mb) carried more W2014 segments than other chromosomal regions. The total size of the introgressed segments detected in the 131 ILs was 2.83 Gb, 7.57 times the total length of the rice genome. The average length of the O. nivara-derived segments carried by each IL was 21.73 Mb, 0.058 times the length of the genome. The average length of single introgressed segment was 3.7 Mb, ranging from 0.17 to 24.7 Mb. Of the introgressed segments, 76.4% were shorter than 5.0 Mb and 9.65% were longer than 10.0 Mb (Fig. 2c). The average length of the homozygous introgressed segments carried by each IL was 4.3 Mb and that of the heterozygous segments was 1.14 Mb (Supplementary Table 2).